Our research concerns the mechanism and consequences of Ty element retrotransposition in the budding yeast Saccharomyces . Ty elements comprise five related families of long terminal repeat (LTR) retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes that correspond to the Gag and Pol genes of retroviruses. The retrotransposon is transcribed into a genome-length RNA, which is the template for reverse transcription by an element-encoded reverse transcriptase protein and for translation. Ty protein maturation and reverse transcription take place within Ty virus-like particles (Ty-VLPs), which appear to be essential for the transposition process. Although Ty-VLPs accumulate in the cytoplasm, a Ty preintegration complex containing Ty cDNA, the element-encoded integrase and perhaps other proteins must transit the nuclear membrane to gain access to the genome. Each Ty element class integrates nonrandomly and possesses distinctive targeting mechanisms that are influenced by the chromatin state or RNA polymerase III transcription factors. All available evidence suggests that Ty elements remain intracellular and are not infectious. Therefore, these elements and their host have evolved control mechanisms to keep transposition and element mediated genome rearrangements at a low level, and integration site preferences that reduce the possibility of causing deleterious mutations. Over the past year, we have made progress on characterizing host genes that modulate Ty1 retrotransposition. The first study involved a systematic screen of 4739 gene-deletion mutants to identify those that increase Ty1 mobility (Ty1 restriction or RTT genes). Among the 91 identified mutants, 80% encode products involved in nuclear processes such as chromatin structure and function, DNA repair and recombination, and transcription. However, bioinformatic analyses encompassing additional Ty1 and Ty3 screens indicate that 264 unique genes involved in a variety of biological processes affect Ty mobility in yeast. Further characterization of 33 of the rtt mutants identified in our screen show that Ty1 RNA levels increase in 5 mutants and the rest affect mobility posttranscriptionally. Ty1 RNA and cDNA levels remain unchanged in mutants defective in transcription elongation, including ckb2&#916; and elf1&#916; , suggesting Ty1 integration may be more efficient in these strains. Insertion site preference at the CAN1 locus requires Ty1 restriction genes involved in histone H2B ubiquitination by Paf complex subunit genes, as well as BRE1 and RAD6 , histone H3 acetylation by RTT109 and ASF1 , and transcription elongation by SPT5 . Our results indicate that multiple pathways restrict Ty1 mobility and histone modifications may protect coding regions from insertional mutagenesis. Since these genes are also required for efficient transcription by RNA polymerase II, additional targets for Ty1 insertion maybe uncovered by stalled transcription complexes. Ongoing work is focused on defining the Ty1 integrase targeting domain and understanding the genomic landscape available for transposition events in wild type and targeting-defective mutants. Considering the large number of genes identified in various screens that modulate Ty retrotransposition, we considered the possibility that many of the RTT genes act through a few common pathways. Support for this idea is evident from a recent study on a subset of Ty1 restriction genes, performed in collaboration with Joan Curcio's laboratory (Wadsworth Center, Albany NY). Mobility of Ty1 in budding yeast is restricted by an array of proteins that function to preserve the integrity of the genome during DNA replication and repair. However, the mechanisms involved in increasing Ty1 cDNA levels and mobility in the absence of these Rtt factors, several of which are orthologs of mammalian retroviral restriction factors, are poorly characterized. Interestingly, two S-phase checkpoint pathways, the replication stress pathway or the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt mutants with defects in genome preservation. In contrast, neither checkpoint pathway is involved in activating Ty1 in two rtt mutants that are competent for genome maintenance. In rtt101&#8710; mutants, in which elevated transposition is stimulated through DNA damage checkpoints proteins, Rad9, Rad24, Mec1, Rad53 and Dun1 but not Chk1, Ty1-encoded proteins, rather than Ty1 cDNA, are the direct targets of the checkpoint pathway. Levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated in rtt101&#8710; mutants. We hypothesize that DNA lesions created in the absence of genome integrity factors function as triggers that enhance Ty1 reverse transcriptase activity via S-phase checkpoint pathways.